Abstract It is widely accepted that cell-type-specific gene expression is primarily achieved by cell-type-specific presence of transcription factors (TFs), which bind to cognate DNA sequences. TFs then initiate changes in higher-order chromatin structures by recruiting chromatin modifiers, including histone-modifying enzymes. Unlike TFs, chromatin modifiers tend to be ubiquitously expressed. Among the plethora of chromatin modifications, regulators of histone methylation are more frequently mutated in neurodevelopmental disorders (NDDs) such as intellectual disabilities (IDs) and autism. Why is the brain so sensitive to dysregulation of histone methylation? Is methyl-histone regulation in neurons unique? Investigation of a limited number of cell types, cancer-cell lines, and embryonic stem cells has hampered our ability to address these questions. The overarching goal of my research group is to contribute to the understanding of how methyl-histone regulations underlie normal and pathological brain functions. Our focus is on the LSD1-PHF21A histone- demethylation complex, which involves neuron-specific alternative splicing. LSD1 is a histone demethylase for histone H3 lysine 4 (H3K4me). PHF21A was the first-discovered ?zero reader,? which recognizes unmethylated H3K4 (H3K4me0), the reaction product of canonical LSD1 (LSD1-c). Both LSD1 and PHF21A haploinsufficiencies lead to NDDs, suggesting their importance in brain development. The neuronal LSD1 isoform (LSD1-n), which carries an alternative exon in its catalytic domain, was reported to have distinct substrate specificity. However, the specific lysine(s) targeted by LSD1-n remains controversial. The goal of this proposal is to determine the roles of the neuronal LSD1-PHF21A complex. Our preliminary study showed that PHF21A also carries an alternative exon right upstream of the H3K4me0-recognizing PHD finger. This region of PHF21A contains an AT-hook motif, which directly binds to DNA; we found that the alternative exon disrupts the AT-hook, hence the DNA binding, but not H3K4me0 binding. These observations raise an exciting possibility that the neuronal PHF21A isoform (PHF21A-n) recognizes nucleosomes in a distinct manner compared to canonical PHF21A (PHF21A-c), thereby cooperating with LSD1-n to generate the neuronal transcriptome for normal brain development. We propose testing the hypothesis using multidisciplinary approaches encompassing cell biology, biochemistry, and structural biology. The research plan was developed to provide both mechanistic insights into the regulation of histone modifications and a better understanding of the pathogenesis of neurodevelopment disorders, which could lead to novel approaches for brain-specific therapeutic targets.